An ever-evolving animal health emergency

A form of highly pathogenic avian influenza (HPAI) has caused widespread damage and disease in birds since it was first detected about a year ago. Last March, a bald eagle found dead in Pennsylvania’s Chester County became the first confirmed case in the state.

Since then, through the Wildlife Futures Program and the Pennsylvania Animal Diagnostic Laboratory System (PADLS), Penn’s School of Veterinary Medicine has stepped up its diagnostic work related to HPAI, supporting agencies including the Pennsylvania Game Commission and the Department of Agriculture of the United States in monitoring the disease in both wild and domestic birds.

The outbreak, caused by the H5N1 HPAI strain, has passed a milestone: more than 50.5 million birds affected. The figure surpasses that of a 2014-15 HPAI outbreak, which at the time was considered “the largest animal health emergency in US history based on the number of birds affected,” says Lisa Murphy, who directs the PADLS laboratory at Bolton Center’s Penn Vet’s New Campus.

While avian flu viruses typically burn out during the summer as wild birds finish their migrations, this outbreak roared back in the fall and is now in 46 states, with Pennsylvania suffering particularly severe losses of more than 4 million of birds. Why remains an open question, but new lines of research by Penn Vet researchers, including Eman Anis, a microbiologist, and Louise Moncla, who joined the faculty in September, aim to understand, at the genetic level, the reasons why which this particular virus is causing so much damage.

Tracking a virus through testing

Even when there isn’t an active disease outbreak, PADLS routinely tests for bird flu as part of statewide surveillance efforts. “We’re available every weekday and on call during the weekends,” says Anis, an assistant professor of microbiology.

Penn Vet’s New Bolton Center PADLS laboratory processes samples from birds suspected of having bird flu. Of the 368 wild bird samples tested between late January and September this year, 53 tested positive for HPAI H5N1. (Image: Courtesy of PADLS and the Pennsylvania Game Commission)

Last winter, even before this strain of the virus hit Pennsylvania, the lab began stocking up on supplies, enough to run at least 500 samples a day. “And while we typically only test samples from domestic poultry, we have received samples from wild birds through our Wildlife Futures program,” Murphy says, including the bald eagle that scored the first confirmed case of HPAI in the state. Specimens suspected of being HPAI positive are sent for confirmatory testing to the National Veterinary Services Laboratory.

Testing at PADLS takes place in a secure biosafety level 2 laboratory at the New Bolton Center and involves a PCR test, short for polymerase chain reaction, which looks for particular sequences in the genome that are characteristic of some viruses. If that test reveals the presence of influenza A virus, the group to which HPAI belongs, further PCR testing can determine the subtype.

Viruses that are found to possess a hemagglutinin (H) protein 5 or 7 undergo additional testing to determine whether they are highly or low pathogenic. This distinction has to do with how the virus’s hemagglutinin protein attaches itself to host cells. Mutations that give the virus the ability to bind to more host cell types and to spread and infect cells more easily are likely to cause more serious disease.

While low pathogenic influenza viruses can cause mild symptoms that may be missed, highly pathogenic viruses can affect multiple organ systems. “Some affected birds have neurological signs, so they may have difficulty flying or appear disoriented,” Anis says.

When a so-called “high path” virus is found in a commercial flock, the entire flock typically has to cull its birds. This happened to three operators in Pennsylvania’s Lehigh Valley before Thanksgiving, the farms killed more than 60,000 birds “at the worst possible time,” says Murphy.

How viruses evolve, spread and wreak havoc

Penn Vet’s PADLS Laboratory recently requested and received permission from the US Department of Agriculture and Penn Biosafety Regulators to retain non-infectious genetic material from samples they test for HPAI. Researchers, including Anis and Moncla, plan to start analyzing this RNA soon to understand the virus’s evolution and ability to cross-species.

Moncla will rely in part on the digital tools it has curated to help visualize these transmission patterns and pathways for a variety of pathogens, including the H5N1 avian virus. An open source platform she started working on during her postdoctoral fellowship called Nextstrain allows users to monitor what is known about the relationship between detected pathogens. The data there informs a phylogenetic tree of H5N1 – in other words, the virus family tree – indicating that the strain currently circulating in the US originated in China, spread to Southeast Asia and then Europe , before moving to North America.

Seven Turkey Vultures perch on the bare branches of a tree against

A feature of the current epidemic that distinguishes it from previous ones is the persistent transmission in wild birds. Birds of prey, like these turkey vultures, seem especially vulnerable to infection.

What isn’t clear is why the virus is behaving so differently from previous strains of bird flu. The strain, like most H5N1 HPAIs, does not typically target people; only two human cases have been confirmed, both in individuals who work closely with birds. However, while some avian influenza viruses are somewhat limited in the species they affect, this H5N1 virus appears to be less fussy.

Infections have been found in a wide range of birds and some mammals, including foxes, bears and even seals. The scientists also observed extensive transmission of the disease within wild bird populations that has persisted beyond the spring migration season and is intensifying again with the autumn migration.

“Traditionally, wild waterfowl, such as ducks and geese, were thought to be the primary concern when it came to transmission,” says Murphy. “But with this, we’ve had a lot of problems with birds of prey, birds of prey like bald eagles and vultures. With vultures in particular they roost in large groups, so you can imagine the virus spreading rapidly through a population.

The strain’s apparent ability to affect wild birds and to move between wild and domesticated populations, Moncla says, could be due to particular genetic variations that allow the protein hemagglutinin to bind to a wider than normal range of cells of the host species, or there may be other factors at play. Just what is responsible is what he hopes to discover by sequencing the virus in his own laboratory.

“There is currently no publicly available avian flu genomics data from Pennsylvania, despite the fact that there is a large outbreak here,” Moncla says. “My goal would be to find actionable information: How does this virus travel between domestic and wild birds in the state? Are there particular species that act as sources of new genetic diversity? I think we have an opportunity to be at the forefront of watching this virus as it becomes endemic in North America.”

Eman Anis is an assistant professor of microbiology in the Department of Pathobiology and is the section head of the Pennsylvania Animal Diagnostic Laboratory System (PADLS) New Bolton Center Molecular Laboratory at Penn’s School of Veterinary Medicine.

Louise Moncla is an assistant professor of pathobiology at Penn’s School of Veterinary Medicine.

Lisa Murphy is the resident director of PADLS New Bolton Center, professor of toxicology, associate director of the Institute for Infectious and Zoonotic Diseases, and co-director of the Wildlife Futures Program at Penn’s School of Veterinary Medicine.

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